The aminoacyl-tRNA synthetases arose early in evolution and established the rules of the genetic code through their specific interactions with amino acids and RNA molecules. About half of these tRNA charging enzymes are class I synthetases, which contain similar N-terminal nucleotide-fold-like structures that are joined to variable domains implicated in specific protein-tRNA contacts. Here, we show that a bacterial synthetase gene can be split into two nonoverlapping segments. We split the gene for Escherichia coli methionyl-tRNA synthetase (a class I synthetase) at several sites near the interdomain junction, such that one segment codes for the nucleotide-fold-containing domain and the other provides determinants for tRNA recognition. When the segments are folded together, they can recognize and charge tRNA, both in vivo and in vitro. We postulate that an early step in the assembly of systems to attach amino acids to specific RNA molecules may have involved specific interactions between discrete proteins that is reflected in the interdomain contacts of modern synthetases.